Oxazole ring-containing C30 terpenoid polymer compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a C30 terpenoid polymer compound containing an oxazole ring as well as a preparation method and application of the C30 terpenoid polymer compound. According to the method, various separation technologies and means are utilized, pond fir seeds are taken as a separation target, enrichment of the terpenoid polymer is realized by utilizing a molecular sieve principle of gel, then silica gel column chromatography and ODS column chromatography separation are sequentially performed to obtain a target fraction, reversed-phase high-performance liquid chromatography purification and normal-phase chiral column chromatography resolution are sequentially performed, and the terpenoid polymer is obtained. The C30 terpenoid polymer compounds as shown in the formula I and the formula II are obtained. In-vitro anti-tumor cell experiments prove that the C30 terpenoid polymer compound has remarkable anti-tumor activity, can be used as an active ingredient of an anti-tumor drug, and has wide application. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology and relates to a C 30 Terpene polymer compounds and preparation methods and applications thereof. Background Art

[0002] Terpene polymers are a relatively rare type of terpene polymers formed by the polymerization of monomeric terpenes. They are widely distributed in a variety of medicinal plants such as Taxaceae, Leguminosae, and Lamiaceae. Common terpene polymers are mostly composed of sesquiterpenes and sesquiterpenes, sesquiterpenes and diterpenes, monoterpenes and diterpenes, diterpenes and diterpenes, diterpenes and triterpenes through different linking methods such as DA cyclization, [2+2], [5+2], CC single bonds, ether bonds, and ester bonds to form complex and variable skeleton types, thereby showing diverse and significant biological activities. The C 30 Terpene polymers are a relatively rare class of polymers. Modern pharmacological studies have shown that C 30 Terpenoid polymers exhibit multiple activities such as anti-tumor, anti-inflammatory, anti-tumor cell migration, inhibition of non-alcoholic fatty liver disease, and hypoglycemic activity. Monoterpenes and diterpenes connected by oxazole rings are a new class of terpenoid polymers that have not been reported before, and are also characteristic components of the Taxaceae family. Studies have found that terpenoid polymers found in the Taxaceae family have significant therapeutic potential for cancer, diabetes, neuroprotection and other diseases.

[0003] In terms of chemical structure, C 30 Terpene polymers are polymers formed by connecting diterpenes and monoterpenes through oxazole rings. They are a relatively rare type of terpene polymers. Due to the novelty of their structure and significant biological activity, they are increasingly attracting the interest of scientific researchers. Due to the particularity of this type of structure, although it is a terpene containing thirty carbon atoms, it is not a terpene composed of five isoprenes connected end to end as defined by triterpenes, but rather a rosinane-type diterpene unit connected to a monoterpene through an oxazole ring to form a C 30 A terpene polymer having a hydroxyethyl substitution at the 7-position. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a C containing an oxazole ring 30 Terpene polymer compounds and preparation methods and applications thereof.

[0005] To solve the above problems, the technical solution provided by the present invention is as follows:

[0006] In the first aspect of the present invention, the present invention provides a C containing an oxazole ring 30 The terpene polymer compound is characterized in that its structural formula is as shown in Formula I or Formula II:

[0007]

[0008] In the second aspect of the present invention, the present invention provides a C containing an oxazole ring as described above. 30 The preparation method of terpene polymer compounds comprises the following steps: isolating the C containing oxazole ring from the mature seeds of pond cypress 30 Terpene polymer compounds.

[0009] As the C containing oxazole ring of the present invention 30 A preferred embodiment of the method for preparing terpene polymer compounds comprises the following steps:

[0010] S1. The mature seeds of the pond cypress are extracted with a first organic solvent, and the obtained extract is concentrated in vacuo to be alcohol-free to obtain an extract;

[0011] S2. suspending the extract in water and extracting with a second organic solvent to obtain an extract;

[0012] S3. Separating the extract by column chromatography to obtain the C containing oxazole ring 30 Terpene polymer compounds.

[0013] As the C containing oxazole ring of the present invention 30 In a preferred embodiment of the method for preparing terpene polymer compounds, the first organic solvent is ethanol, preferably 95% ethanol; and the second organic solvent is ethyl acetate.

[0014] As the C containing oxazole ring of the present invention 30 In a preferred embodiment of the method for preparing terpene polymer compounds, step S3 comprises the following steps:

[0015] S31. The extract is separated by gel column chromatography to obtain an enriched portion of the terpene polymer;

[0016] S32. The enriched part is sequentially subjected to silica gel column chromatography and ODS column chromatography to obtain a target fraction;

[0017] S33. The target fraction is purified by reverse phase high performance liquid chromatography and separated by normal phase chiral column chromatography to obtain C shown in formula I and formula II. 30 Terpene polymer compounds.

[0018] As the C containing oxazole ring of the present invention 30 In a preferred embodiment of the method for preparing terpene polymer compounds, the gel column chromatography uses methanol as the elution solvent, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution, and the ODS column chromatography uses a methanol / water mixed solvent for gradient elution.

[0019] As the C containing oxazole ring of the present invention 30 A preferred embodiment of the preparation method of terpene polymer compounds is that the reversed-phase high performance liquid chromatography uses an XB-C18 chromatographic column with a specification of 5μm, 21.2×250mm, and the mobile phase of the reversed-phase high performance liquid chromatography is a methanol-water solution, in which the volume ratio of methanol to water is 95:5.

[0020] In the third aspect of the present invention, the present invention also provides a C containing an oxazole ring as described above. 30 Application of terpenoid polymer compounds in the preparation of anti-tumor drugs.

[0021] The above-mentioned C containing oxazole ring 30 Terpene polymer compounds include C shown in formula I and / or formula II 30 Terpene polymer compounds.

[0022] In the fourth aspect of the present invention, the present invention also provides a drug, the drug comprising the above-mentioned C containing oxazole ring 30 A terpene polymer compound or a pharmaceutically acceptable salt thereof.

[0023] In some embodiments, the above-mentioned medicine further includes pharmaceutically acceptable excipients and carriers.

[0024] In some embodiments, the pharmaceutical preparation of the above-mentioned drug is granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels or injections.

[0025] Compared with the prior art, the present invention selects mature pond cypress seeds, performs reflux extraction with ethanol, extracts with ethyl acetate multiple times, and then uses various separation techniques and means to obtain the above-mentioned terpene polymers containing oxazole rings of formula I to formula II. The C 30 Terpene polymers are a new type of terpene polymers discovered for the first time. The discovery of this type of novel structure enriches the skeleton and types of terpene polymers, and also provides ideas and sources for the discovery of natural drugs. They have significant anti-tumor activity and can be used as active ingredients of anti-tumor drugs, providing alternative compounds for the development of new anti-tumor drugs. DETAILED DESCRIPTION

[0026] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or the product specifications are carried out. Where specific conditions are not specified in the following embodiments, the conditions are carried out according to conventional conditions or manufacturer recommendations, and the methods used are conventional methods known in the art if not otherwise specified, and the consumables and reagents used are commercially available if not otherwise specified. Unless otherwise indicated, the professional and scientific terms used herein are identical in meaning to those skilled in the art. In addition, any method or material similar or equal to the recorded content may also be applied to the present invention.

[0027] Example 1: Preparation of compounds of formula I-II

[0028] The present invention uses mature seeds of Taxodium truncatum of the Taxodiaceae family to perform reflux extraction with 95% ethanol, and prepares the compound of the present invention through the steps of extraction, enrichment, separation, etc. The seeds of Taxodium truncatum are collected from Nantong, Jiangsu.

[0029] Taking the seeds of pond cypress as an example, the process of preparing the compounds of formula I-II is as follows:

[0030] 1) Extraction, enrichment and initial separation

[0031] The mature and dried seeds of the pond cypress were crushed (10 kg), refluxed and extracted three times with 95% ethanol, and then vacuum concentrated to alcohol-free, mixed with hot water (60°C), and then extracted multiple times with ethyl acetate in equal proportions. The extract layer (0.5 kg) was subjected to gel column chromatography, using methanol as the elution solvent, and each 200 mL was a fraction to obtain fraction Fr.1-8. After LC-MS identification, the Fr.1-3 fractions were combined and separated by silica gel column chromatography. The elution solvents were successively petroleum ether / ethyl acetate with a volume ratio of 100:0, petroleum ether / ethyl acetate with a volume ratio of 100:5, petroleum ether / ethyl acetate with a volume ratio of 100:10, petroleum ether / ethyl acetate with a volume ratio of 100:20, petroleum ether / ethyl acetate with a volume ratio of 100:30, petroleum ether / ethyl acetate with a volume ratio of 100:50, petroleum ether / ethyl acetate with a volume ratio of 100:100, and petroleum ether / ethyl acetate with a volume ratio of 0:100, to obtain eight fractions Fr.AH, and each fraction was detected by LC-MS, and the fraction Fr.C was locked.

[0032] 2) ODS-C18 column chromatography separation

[0033] The fraction Fr.C was separated by ODS-C18 column chromatography. The fractions were separated by methanol / water with a volume ratio of 60:40, methanol / water with a volume ratio of 70:30, methanol / water with a volume ratio of 80:20, methanol / water with a volume ratio of 85:15, methanol / water with a volume ratio of 90:10, methanol / water with a volume ratio of 95:5, and methanol / water with a volume ratio of 100:0. The fractions were collected, namely Fr.C1-Fr.C7, and after HPLC and LC-MS analysis, the target fraction Fr.C5 was locked.

[0034] 3) Reverse-phase HPLC purification and chiral column separation

[0035] The fraction Fr.C5 was purified by reverse phase high performance liquid chromatography. The preparation conditions are as follows: XB-C18 (5μm, 21.2×250mm), methanol-water (95:5, V / V), flow rate 10.0mL / min, detection wavelength 230nm, and NMR data showed a 1:1 and very similar mixture of formula I and formula II. The chiral column was used to separate formula I and formula II. The separation conditions were Daicel AD-H (5μm, 10.0×250mm), n-hexane-isopropanol (98:2), and the detection wavelength was 230nm. The mass spectrometry and spectroscopy data of formula I-formula II are shown below.

[0036] Compound Ⅰ: taxodascaloid A; optical rotation (c 0.18MeOH); UV absorption (MeOH)λ max (logε)215(3.58)nm; main absorption peak of infrared spectrum (KBr)ν max 2923, 2853, and 1659cm -1 ; High resolution mass spectrum m / z is 494.3626 [M+H] + (calcd for C 32 H 48 NO3), the H NMR and C NMR data are shown in Table 1.

[0037] Compound II: taxodascaloid B; optical rotation (c 0.12MeOH); UV absorption (MeOH)λ max (logε)205(3.77)nm; main absorption peak of infrared spectrum (KBr)ν max 2922, 2851, and 1658cm -1 ; High resolution mass spectrum m / z is 494.3640 [M+H] + (calcd for C 32 H 48 NO3), the H NMR and C NMR data are shown in Table 1.

[0038] Table 1. H NMR and C NMR data of compounds of formula I to II

[0039]

[0040]

[0041] The above results show that the structures of the obtained compounds of formula I to formula II are correct.

[0042] Example 2: Antitumor activity of compounds of formula I-II

[0043] 1) Experimental Materials

[0044] Instruments and reagents: CO2 incubator (Thermo Fisher Scientific, USA); CKX41 microscope (Olympus Optical Industry Co., Ltd., Japan); Easy pure II laboratory-grade ultrapure water instrument (Thermo Fisher Scientific, USA); DMSO (Biosharp, USA); DMEM / RPMI 1640 culture medium (Invitrogen, USA); DMSO, MTT, trypsin and cisplatin, etc. (Sigma, USA); fetal bovine serum (Hangzhou Sijiqing Biomaterials Research Institute); other commonly used inorganic salts and other reagents are all analytical grade (Nanjing Chemical Reagent Co., Ltd.).

[0045] The tumor cell lines used for the test: HCT-116 cells (colon cancer cells), MDA-MB-231 cells (human breast cancer cells), A549 cells (non-small cell lung cancer cell line), were purchased from the Institute of Oncology, Chinese Academy of Medical Sciences.

[0046] Test sample: C from pond cypress seeds 30 The terpene polymer compounds of formula I and formula II have a purity of more than 95%. Cisplatin is selected as a positive control drug, and each compound is dissolved in DMSO and then diluted.

[0047] 2) Experimental methods

[0048] The MTT method was used to determine the half inhibition rate IC of each test compound on tumor cell lines. 50 Value: Tumor cells in the logarithmic growth phase were taken and the cell concentration was adjusted to 5×10 5 / mL, inoculated in 96-well culture plates, 100 μL of cell suspension was added to each well of the drug-treated group and the cell control group, and 3 replicates were set up for each group. Only RPMI 1640 complete culture medium was added to the blank control group, 100 μL per well, and 3 replicates were set up. After the 96-well culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours, different concentrations of the test samples were added to make the final concentrations of 0, 10, 20, and 40 μM, and the culture was continued for 24 hours. The absorbance (A) value at 570 nm was measured on a microplate reader according to the MTT method, and the inhibition rate was calculated [inhibition rate = (1-experimental group A value / control group A value) × 100%]. The experiment was repeated 3 times. SPSS11.5 software was used to make a regression equation to calculate the half-maximal inhibitory concentration (IC) of each test sample on tumor cells for 24 hours. 50 ).

[0049] 3) Experimental results

[0050] According to the MTT test results, calculate the IC of the test sample for the above cells 50 The results are shown in Table 2.

[0051] Table 2. In vitro cytotoxic activity screening results of test samples

[0052]

[0053] a The above controls and samples were repeated three times.

[0054] The results show that the compounds of formula I and formula II of the present invention have good anti-tumor activity and can be used as active ingredients of anti-tumor drugs.

[0055] Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A C containing an oxazole ring 30 The terpene polymer compound is characterized in that: Its structural formula is shown in Formula I or Formula II:

2. A C containing an oxazole ring as claimed in claim 1 30 The method for preparing terpene polymer compounds is characterized in that: The preparation method comprises: isolating the C containing oxazole ring from the mature seeds of Pool Cypress 30 Terpene polymer compounds.

3. The preparation method according to claim 2, characterized in that: The steps include: S1. The mature seeds of the pond cypress are extracted with a first organic solvent, and the obtained extract is concentrated in vacuo to be alcohol-free to obtain an extract; S2. suspending the extract in water and extracting with a second organic solvent to obtain an extract; S3. Separating the extract by column chromatography to obtain the C containing oxazole ring 30 Terpene polymer compounds.

4. The preparation method according to claim 3, characterized in that: The first organic solvent is ethanol, and the second organic solvent is ethyl acetate.

5. The preparation method according to claim 3, characterized in that: Step S3 specifically includes the following steps: S31. The extract is separated by gel column chromatography to obtain an enriched portion of the terpene polymer; S32. The enriched part is sequentially subjected to silica gel column chromatography and ODS column chromatography to obtain a target fraction; S33. The target fraction is purified by reverse phase high performance liquid chromatography and separated by normal phase chiral column chromatography to obtain C shown in formula I and formula II. 30 Terpene polymer compounds.

6. The preparation method according to claim 5, characterized in that: The gel column chromatography uses methanol as an elution solvent, the silica gel column chromatography uses a petroleum ether / ethyl acetate mixed solvent for gradient elution, and the ODS column chromatography uses a methanol / water mixed solvent for gradient elution.

7. The preparation method according to claim 5, characterized in that: The reversed-phase high performance liquid chromatography uses an XB-C18 chromatographic column with a specification of 5 μm, 21.2×250 mm. The mobile phase of the reversed-phase high performance liquid chromatography is a methanol-water solution, in which the volume ratio of methanol to water is 95:

5.

8. A C containing an oxazole ring as claimed in claim 1 30 Application of terpenoid polymer compounds in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The C containing an oxazole ring 30 Terpene polymer compounds include C shown in formula I and / or formula II 30 Terpene polymer compounds.

10. A drug, characterized in that The drug comprises the C containing oxazole ring according to claim 1 30 A terpene polymer compound or a pharmaceutically acceptable salt thereof.

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